Quantitative PCR integrates PCR amplification of template cDNAs with flourescence detection, recording accumulation of amplicons cycle-by-cycle (1). A known qPCR template standard serves two main purposes. It functions as a positive control and as a reference for measuring the exact copy number of a transcript in an unknown sample(1). Quantitative PCR (QPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models, in serum and in tissue samples, and both for culturable viruses and non-culturable viruses(2). Apart from increased sensitivity, this technique offers a high-throughput alternative to Southern blots for determining gene copy number, a significant factor when screening fungal mutants and transformants (3).
qPCR protocol: Preparation of the qPCR template standard 1. The qPCR template standard is provided as a dried pellet. Add 50 μl of d H2O to the tube and
vortex briefly. The resuspended template is 10,000,000 copy/μl. Thaw the 10 x dilution buffer
tube from the kit at room temperature.
2. If the SYBR Green I method is used, reconstitute SybGREEN primer by adding 200 μl d H2O to
the primer tube and vortex it briefly. The resuspended primers are 10 pmol/ul each. (Omit this
step if Taqman probe method is used)
3. Prepare 1.0 mL of 1 x dilution buffer by adding 100 μl of the 10 X buffer to 900 μl H2O.
4. Label six (6) Eppendorf tubes from 1 to 6. Transfer 90 μl of 1x buffer to each pre-labeled
eppendorf tube (1 to 6).
5. Transfer 10 μl of template standard from the stock tube to eppendorf tube #1 (Fig. 2). Mix the
solution by briefly vortexing.
6. Transfer 10 μl solution from tube #1 to tube #2, then mix the solution by vortexing..
7. Perform remaining dilutions by repeating steps 5 and 6.
8. Transfer 5 μl of diluted template solution from each tube to a 96-well PCR plate (use 2 μl when a
384-well plate is used).
Their are two basic options when choosing reporter fluorophore: The first, simplest and least expensive is the intercalating dye SYBR Green I, the fluorescence of which is very low in the absence of double-stranded DNA and very high in its presence. The second and more sensitive option comprises one of a collection of fluorophore-labeled oligonucleotide probes (e.g., Taqman, Molecular Beacons or Scorpion probes), designed to anneal near the middle of an accumulating amplicon. Probes contain a reporter fluorophore, e.g., 6-FAM, typically at the 5' end, and a quenching fluorophore or "black-hole" (non-fluorescing) dye at the 3' end (1).
Works Cited
1. Bustin S.A. (2002). Quantification of mRNA using real-time reverse transcription PCR: trends and problems. Journal of Molecular Endocrinology 29, 23-39.
2. Crotty Shane, McCausland Megan M. (2008). Quantitative PCR technique for detecting lymphocytic choriomeningitis virus in vivo. J Virol Methods 147 (1), 167-176.
3. Solomon Peter, Simon V.S. Ipcho, James K. Hane, Kar-Chun Tan, Richard P. Oliver (2007). A quantitative PCR approach to determine gene copy number. Fungal genetic reports 55, 5-8.
qPCR protocol: Preparation of the qPCR template standard
1. The qPCR template standard is provided as a dried pellet. Add 50 μl of d H2O to the tube and
vortex briefly. The resuspended template is 10,000,000 copy/μl. Thaw the 10 x dilution buffer
tube from the kit at room temperature.
2. If the SYBR Green I method is used, reconstitute SybGREEN primer by adding 200 μl d H2O to
the primer tube and vortex it briefly. The resuspended primers are 10 pmol/ul each. (Omit this
step if Taqman probe method is used)
3. Prepare 1.0 mL of 1 x dilution buffer by adding 100 μl of the 10 X buffer to 900 μl H2O.
4. Label six (6) Eppendorf tubes from 1 to 6. Transfer 90 μl of 1x buffer to each pre-labeled
eppendorf tube (1 to 6).
5. Transfer 10 μl of template standard from the stock tube to eppendorf tube #1 (Fig. 2). Mix the
solution by briefly vortexing.
6. Transfer 10 μl solution from tube #1 to tube #2, then mix the solution by vortexing..
7. Perform remaining dilutions by repeating steps 5 and 6.
8. Transfer 5 μl of diluted template solution from each tube to a 96-well PCR plate (use 2 μl when a
384-well plate is used).
Their are two basic options when choosing reporter fluorophore: The first, simplest and least expensive is the intercalating dye SYBR Green I, the fluorescence of which is very low in the absence of double-stranded DNA and very high in its presence. The second and more sensitive option comprises one of a collection of fluorophore-labeled oligonucleotide probes (e.g., Taqman, Molecular Beacons or Scorpion probes), designed to anneal near the middle of an accumulating amplicon. Probes contain a reporter fluorophore, e.g., 6-FAM, typically at the 5' end, and a quenching fluorophore or "black-hole" (non-fluorescing) dye at the 3' end (1).
Works Cited
1. Bustin S.A. (2002). Quantification of mRNA using real-time reverse transcription PCR: trends and problems. Journal of Molecular Endocrinology 29, 23-39.
2. Crotty Shane, McCausland Megan M. (2008). Quantitative PCR technique for detecting lymphocytic choriomeningitis virus in vivo. J Virol Methods 147 (1), 167-176.
3. Solomon Peter, Simon V.S. Ipcho, James K. Hane, Kar-Chun Tan, Richard P. Oliver (2007). A quantitative PCR approach to determine gene copy number. Fungal genetic reports 55, 5-8.